Expansion of the Spore Surface Polysaccharide Layer in Bacillus subtilis by Deletion of Genes Encoding Glycosyltransferases and Glucose Modification Enzymes

Bentley Shuster, Mark Khemmani, Yusei Nakaya, Gudrun Holland, Keito Iwamoto, Kimihiro Abe, Daisuke Imamura, Nina Maryn, Adam Driks, Tsutomu Sato, Patrick Eichenberger

Research output: Contribution to journalArticle

Abstract

Polysaccharides (PS) decorate the surface of dormant endospores (spores). In the model organism for sporulation, Bacillus subtilis, the composition of the spore PS is not known in detail. Here, we have assessed how PS synthesis enzymes produced during the late stages of sporulation affect spore surface properties. Using four methods, bacterial adhesion to hydrocarbons (BATH) assays, India ink staining, transmission electron microscopy (TEM) with ruthenium red staining, and scanning electron microscopy (SEM), we characterized the contributions of four sporulation gene clusters, spsABCDEFGHIJKL, yfnHGF-yfnED, ytdA-ytcABC, and cgeAB-cgeCDE, on the morphology and properties of the crust, the outermost spore layer. Our results show that all mutations in the sps operon result in the production of spores that are more hydrophobic and lack a visible crust, presumably because of reduced PS deposition, while mutations in cgeD and the yfnH-D cluster noticeably expand the PS layer. In addition, yfnH-D mutant spores exhibit a crust with an unusual weblike morphology. The hydrophobic phenotype from sps mutant spores was partially rescued by a second mutation inactivating any gene in the yfnHGF operon. While spsI, yfnH, and ytdA are paralogous genes, all encoding glucose-1-phosphate nucleotidyltransferases, each paralog appears to contribute in a distinct manner to the spore PS. Our data are consistent with the possibility that each gene cluster is responsible for the production of its own respective deoxyhexose. In summary, we found that disruptions to the PS layer modify spore surface hydrophobicity and that there are multiple saccharide synthesis pathways involved in spore surface properties.IMPORTANCE Many bacteria are characterized by their ability to form highly resistant spores. The dormant spore state allows these species to survive even the harshest treatments with antimicrobial agents. Spore surface properties are particularly relevant because they influence spore dispersal in various habitats from natural to human-made environments. The spore surface in Bacillus subtilis (crust) is composed of a combination of proteins and polysaccharides. By inactivating the enzymes responsible for the synthesis of spore polysaccharides, we can assess how spore surface properties such as hydrophobicity are modulated by the addition of specific carbohydrates. Our findings indicate that several sporulation gene clusters are responsible for the assembly and allocation of surface polysaccharides. Similar mechanisms could be modulating the dispersal of infectious spore-forming bacteria.

Original languageEnglish (US)
JournalJournal of bacteriology
Volume201
Issue number19
DOIs
StatePublished - Oct 1 2019

Fingerprint

Glycosyltransferases
Gene Deletion
Bacillus subtilis
Spores
Polysaccharides
Glucose
Enzymes
Surface Properties
Multigene Family
Operon
Hydrophobic and Hydrophilic Interactions
Mutation
Nucleotidyltransferases
Staining and Labeling
Bacterial Adhesion
Bacteria
Ruthenium Red

Keywords

  • Bacillus subtilis
  • cell surface
  • polysaccharides
  • spore coat
  • spore crust
  • sporulation

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology

Cite this

Expansion of the Spore Surface Polysaccharide Layer in Bacillus subtilis by Deletion of Genes Encoding Glycosyltransferases and Glucose Modification Enzymes. / Shuster, Bentley; Khemmani, Mark; Nakaya, Yusei; Holland, Gudrun; Iwamoto, Keito; Abe, Kimihiro; Imamura, Daisuke; Maryn, Nina; Driks, Adam; Sato, Tsutomu; Eichenberger, Patrick.

In: Journal of bacteriology, Vol. 201, No. 19, 01.10.2019.

Research output: Contribution to journalArticle

Shuster, Bentley ; Khemmani, Mark ; Nakaya, Yusei ; Holland, Gudrun ; Iwamoto, Keito ; Abe, Kimihiro ; Imamura, Daisuke ; Maryn, Nina ; Driks, Adam ; Sato, Tsutomu ; Eichenberger, Patrick. / Expansion of the Spore Surface Polysaccharide Layer in Bacillus subtilis by Deletion of Genes Encoding Glycosyltransferases and Glucose Modification Enzymes. In: Journal of bacteriology. 2019 ; Vol. 201, No. 19.
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AU - Nakaya, Yusei

AU - Holland, Gudrun

AU - Iwamoto, Keito

AU - Abe, Kimihiro

AU - Imamura, Daisuke

AU - Maryn, Nina

AU - Driks, Adam

AU - Sato, Tsutomu

AU - Eichenberger, Patrick

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N2 - Polysaccharides (PS) decorate the surface of dormant endospores (spores). In the model organism for sporulation, Bacillus subtilis, the composition of the spore PS is not known in detail. Here, we have assessed how PS synthesis enzymes produced during the late stages of sporulation affect spore surface properties. Using four methods, bacterial adhesion to hydrocarbons (BATH) assays, India ink staining, transmission electron microscopy (TEM) with ruthenium red staining, and scanning electron microscopy (SEM), we characterized the contributions of four sporulation gene clusters, spsABCDEFGHIJKL, yfnHGF-yfnED, ytdA-ytcABC, and cgeAB-cgeCDE, on the morphology and properties of the crust, the outermost spore layer. Our results show that all mutations in the sps operon result in the production of spores that are more hydrophobic and lack a visible crust, presumably because of reduced PS deposition, while mutations in cgeD and the yfnH-D cluster noticeably expand the PS layer. In addition, yfnH-D mutant spores exhibit a crust with an unusual weblike morphology. The hydrophobic phenotype from sps mutant spores was partially rescued by a second mutation inactivating any gene in the yfnHGF operon. While spsI, yfnH, and ytdA are paralogous genes, all encoding glucose-1-phosphate nucleotidyltransferases, each paralog appears to contribute in a distinct manner to the spore PS. Our data are consistent with the possibility that each gene cluster is responsible for the production of its own respective deoxyhexose. In summary, we found that disruptions to the PS layer modify spore surface hydrophobicity and that there are multiple saccharide synthesis pathways involved in spore surface properties.IMPORTANCE Many bacteria are characterized by their ability to form highly resistant spores. The dormant spore state allows these species to survive even the harshest treatments with antimicrobial agents. Spore surface properties are particularly relevant because they influence spore dispersal in various habitats from natural to human-made environments. The spore surface in Bacillus subtilis (crust) is composed of a combination of proteins and polysaccharides. By inactivating the enzymes responsible for the synthesis of spore polysaccharides, we can assess how spore surface properties such as hydrophobicity are modulated by the addition of specific carbohydrates. Our findings indicate that several sporulation gene clusters are responsible for the assembly and allocation of surface polysaccharides. Similar mechanisms could be modulating the dispersal of infectious spore-forming bacteria.

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